A humoral fluid such as blood contains various components, some of which are similar to each other in their molecular weights or physiological properties. Accordingly, the measurement of such components requires high specificity and high sensitivity. Furthermore, in order to evaluate various components for diagnosis of a disease, the measuring procedure must of necessity be simple.
Various methods to detect trace components in blood have been developed, among them enzyme immunoassay which is widely employed because of its sensitivity, specificity and ability to process a large number of samples. However, in the case of conventional enzyme immunoassay, the sensitivity is not always adequate and it is not easy to obtain an exact concentration because of the complicated washing procedures and the transferring of tubes.
Various enzyme immunoassays using conjugates of a ligand or an antibody and an enzyme or a material having an enzyme inhibitory activity have been reported in the literature.
For example in Japanese Patent Application 142466/1982 a method of measuring a biological ligand was reported which comprises, contacting a ligand (1) to be measured with a conjugate of a ligand (2) having an antigenic determinant common to one of the antigenic determinant(s) of the above ligand (1) and biotin or its derivatives capable of reacting with avidin or streptoavidin with an antibody capable of reacting with the above common antigenic determinant in an aqueous solution. Thereafter contacting the above conjugate with avidin, streptoavidin or one of their derivatives capable of reacting with biotin, and measuring the biotin enzyme activity.
Also known is a method of measuring a biological ligand which comprises, contacting a ligand to be measured and an enzyme or a conjugate of an enzyme and a macromolecular substance, with a conjugate of an antibody against the above ligand and an antibody against the above enzyme, or with a conjugate of an antibody against the above ligand, an antibody against the above enzyme and a macromolecular substance in an aqueous solution, and measuring the activity of the enzyme.
Another method of measuring a biological ligand is known in the literature which comprises, allowing to coexist a biologically active composition comprising an immobilization phase of an antibody capable of reacting with the ligand (1) to be measured or a biological ligand (2) capable of reacting with the above antibody and an immobilization phase of a biotin-containing enzyme or a biotin-containing enzyme inhibitor, a water-soluble conjugate of the above ligand (2) or the above antibody and a biotin-containing enzyme inhibitor capable of reacting with the above biotin-containing enzyme or a biotinyl enzyme capable of reacting with the above biotin-containing enzyme inhibitor, and the ligand (1) to be measured in an aqueous solution, and measuring the biotin-containing enzyme activity or the biotinyl enzyme inhibitory activity of the biologically active composition or the aqueous solution.
Also known is a method of measuring a biological ligand which comprises, contacting a ligand (1) to be measured and a conjugate of a ligand (2) having an antigenic determinant being common to one of the antigenic determinant(s) of the ligand (1) and a biotin-containing enzyme inhibitor with an antibody capable of reacting with the common antigenic determinant in an aqueous solution, contacting the above combination with a biotin-containing enzyme, and measuring the biotin-containing enzyme activity.